Development of the protocols described in this paper was supported by grants from the following funding agencies: BBSRC (BB/I026359/1), Fight for Sight (1429 and 1822), The Juvenile Diabetes Research Foundation (2-2003-525), Wellcome Trust (074648/Z/04), British Heart Foundation (PG/11/94/29169), HSC R&#38;D Division (STL/4748/13) and MRC (MC_PC_15026).

<ol>
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Dr. Joanna Kur is now an employee of Medtronic (Twin-Cities, Minnesota, USA), her present work does not conflict with that presented here.

The protocols described above require practice but should be achievable with minimal troubleshooting. On an average day we would obtain 6 - 8 usable arterioles from the isolation and achieve 3 - 4 successful experiments. If problems are encountered, however, there are some steps that can be taken to help improve success rates. Occasionally we have found, particularly when using younger rats (&#60; 8 weeks), that the yield of arterioles can be low. To circumvent this problem, we would suggest centrifuging the retinal tissue (10 - 30 s at 500 x g) between each of the trituration steps in steps 1.12 - 1.14. This often helps to improve the yield of vessels but will also increase the amount of cell debris within the preparation.
When cannulating vessels for pressure myography studies it is important to check the arterioles carefully for any side-branches that may have been cleaved close to the bifurcation site. This represents a common cause of leakage and loss of pressure during experimentation. The main issue that can arise with the [Ca2+]i protocols is the level of dye loading. Poor dye loading leads to low signal-to-noise ratios, while overloading results in disruption of normal Ca2+ homeostasis. To reduce the likelihood of either of these issues, a small aliquot of retinal homogenate can be removed, and isolated vessels checked periodically during the loading protocol. When undertaking patch-clamp experiments, the success rate of gigaseal formation is highly dependent on how well the basal lamina has been digested. When initially testing this protocol or using new lots of enzymes it may be necessary to adjust the concentration/duration of enzyme digestion. Carefully monitoring the separation of the endothelial and smooth muscle layers will ensure sufficient digestion to remove enough basal laminal to gain access. Careful monitoring is also necessary to avoid over-digestion, which can manifest as the vessel begins to constrict. If this occurs, the smooth muscle cells are often too fragile for patch clamp recording. When applying enzymes, it is important to include DNAse I to ensure removal of strands of DNA liberated from damaged cells during the isolation process. DNA fragments are sticky and cause the forceps to adhere to the arteriole during the final stages of the cleaning process (step 4.4), often resulting in vessel loss. Cleaning of vessels is technically difficult and is best performed at high magnification (20X) with gentle sweeping motions of closed forceps of the finest possible tip diameter. Between sweeps, clean the forceps with lab roll.
As highlighted earlier, a key motivation behind the development of the protocols outlined in this manuscript was to better understand why blood flow is disrupted during retinal vascular diseases. Most of our work to date has focused on diabetic eye disease28,33. Arterioles can be isolated from the retinas of experimental rodent models of diabetes using the methods described in section 1. When experimenting on isolated retinal arterioles from diabetic animals, it is important to try to closely replicate the hyperglycemic conditions experienced by the vessels in vivo. For this reason, we would normally raise the D-glucose levels in our isolation and experimental solutions to 25 mM. Thickening of vascular basement membranes is a well-recognized phenomenon in retinal vessels during diabetes34,35. When using animals with prolonged diabetes (&#62; 1 months disease duration), increased enzyme concentrations or digestion times are often needed to enable the application of patch-clamp recording methods.
An important limitation of using ex vivo isolated retinal arterioles to study retinal vascular physiology and pathophysiology is the loss of the surrounding retinal neuropile. Although removal of the retinal glial and neuronal cells enables easy access to the retinal vascular smooth muscle cells for cell physiology studies, the response of the vessels to vasoactive mediators can change dramatically in the absence and presence of retinal tissue. The actions of adenosine tri-phosphate (ATP), for example, provide a good illustration of this point. In isolated rat retinal arterioles, addition of ATP triggers a robust constriction of the vessels36, while in the presence of an intact neuropile, the vessels dilate37. Therefore, wherever possible, we usually try to validate key findings from our isolated arteriole preparations using ex vivo retinal whole-mounts and in vivo measurements of vessel diameter and blood flow16,37. Of note, new methods have recently emerged for studying small arterioles and capillaries in whole perfused porcine retinas ex vivo38,39. Such preparations are likely to greatly improve our understanding of how the retinal neuropile regulates retinal arteriolar and capillary tone and how changes in retinal haemodynamics modulate neuronal activity in the retina.
Although the procedures described in this paper are focused on the use of isolated rat retinal arterioles for understanding arteriolar smooth muscle cell physiology, we are currently developing protocols to also enable the study of endothelial cell function in these vessels. In preliminary work, we have been successful in modifying the enzymatic digestion of retinal arteriolar segments to yield viable endothelial cell tubes that are amenable to Ca2+ imaging and patchclamp recording studies. Cannulation of the isolated arterioles at both ends, enabling intraluminal delivery of drugs, could in the future also enable endothelium-dependent vasodilatory responses to be investigated in these vessels.

Understanding how blood flow is controlled in the retina is an important goal, since abnormal blood flow has been implicated in the pathogenesis of a variety of sight-threatening retinal diseases1,2,3,4. The retinal circulation, which supplies the inner retinal neurons and glial cells, has an end artery arrangement, with all of the blood from the retinal arteries and arterioles passing through the capillaries to the retinal venules and finally veins5. Blood flow in the retina is regulated by the tone of the retinal arteries and arterioles as well as the contractile activity of the pericytes located on the walls of the retinal capillaries and post-capillary venules6,7,8. The control of retinal vascular tone is complex and is modulated by a range of inputs from the circulatory system and surrounding retinal tissue, including blood gases, circulating molecules and hormones, and vasoactive substances released from the retinal vascular endothelium and macroglia9,10,11. The retinal arterioles are the small arterial branches of the retina and are composed of a single layer of vascular smooth muscle cells and an inner lining of longitudinally arranged endothelial cells12,13,14. These vessels form the main site of vascular resistance within the retinal circulation and therefore play an important role in the local control of retinal blood flow. Retinal arterioles regulate capillary blood flow in the retina by dilating or constricting their luminal diameter, mediated by changes in vascular smooth muscle contractility10,15,16. Understanding the molecular mechanisms through which retinal arterioles regulate retinal perfusion therefore requires preparations where the arteriolar smooth muscle cells can be accessed and studied in conditions as close to physiological as possible.
Ex vivo preparations of isolated retinal blood vessels provide access to the vascular smooth muscle cells, whilst still retaining their functionality and interconnectivity with the underlying endothelium. Most studies to date using isolated vessels have focused on large bovine or porcine arterial vessels (60 - 150 &#181;m). These can be mounted in commercially available wire or pressure myograph systems to enable pharmacological interrogation of vascular smooth muscle cell contractile mechanisms17,18. Such preparations have greatly contributed to our knowledge of retinal vascular physiology under normal conditions. Few studies have used retinal arterioles isolated from small laboratory animals as their smaller diameter (~ 8 - 45 &#181;m) prevents their use in conventional myography systems19,20,21,22. An important advantage, however, of using vessels from small laboratory animals is the wide availability of genetically modified, transgenic and retinal disease models. Small laboratory animals are also more amenable for in vivo intervention studies.
Here we describe straightforward protocols for isolating and cannulating rat retinal arterioles for pressure myography experiments. Ca2+ imaging and electrophysiology protocols using these vessels are also detailed. These can provide further insights into the regulation of vascular smooth muscle contractility and blood flow in the retina.

<table><tbody><tr><td>Beakers</td><td>Fisherbrand</td><td>15409083</td><td>Or any equilavent product</td></tr><tr><td>Curved Scissors</td><td>Fisher Scientific</td><td>50-109-3542</td><td>Or any equilavent product</td></tr><tr><td>Disposable plastic pipette/ transfer</td><td>Sarstedt</td><td>86.1174</td><td>6mL is best but other sizes are acceptable; remove tip to widen aperture</td></tr><tr><td>Dissecting microscope</td><td>Brunel Microscopes LTD</td><td></td><td>Or any equilavent product</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #5 14095</td><td>Any equivalent fine forceps</td></tr><tr><td>Pasteur pipette</td><td>Fisherbrand</td><td>11546963</td><td>Fire polished to reduce friction but not enough to narrow the tip</td></tr><tr><td>Petri dish</td><td>Sigma-Aldrich</td><td>P7741</td><td>Or any equilavent 10cm product</td></tr><tr><td>Pipette teat</td><td>Fisherbrand</td><td>12426180</td><td>Or any equilavent product</td></tr><tr><td>Purified water supply</td><td>Merek</td><td>Milli-Q Integral Water Purification System</td><td>Or any equilavent system</td></tr><tr><td>Round bottomed test tube</td><td>Fisherbrand</td><td>14-958-10 B</td><td>5 mL; any equilavent product</td></tr><tr><td>Seratted forceps</td><td>Fisher Scientific</td><td>17-467-230</td><td>Or any equilavent product</td></tr><tr><td>Single edge blades</td><td>Agar Scientific</td><td>T585</td><td>Or any equilavent product</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>184</td><td>Or any pliable surface&amp;nbsp;</td></tr><tr><td>Testtube rack</td><td>Fisherbrand Derlin</td><td>10257963</td><td>Or any equilavent product</td></tr><tr><td>Pressure myography</td><td></td><td></td><td></td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>x2 (or any equilavent product)</td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software&amp;nbsp;</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Analysis plugin</td><td>Myotraker&amp;nbsp;</td><td>https://doi.org/10.1371/journal.pone.0091791.s002</td><td>Custom plugin for imageJ Freely available for download</td></tr><tr><td>Cannulation pipette glass</td><td>World Percision instruments</td><td>TW150F-4</td><td>Or any equilavent product</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fluo-4-AM</td><td>Thermo Fisher Scientific</td><td>F14201</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>Dumont #7 14097</td><td>Any equivalent fine forceps</td></tr><tr><td>Fura-2-AM</td><td>Thermo Fisher Scientific</td><td>F1221</td><td>Make 1 mM stock in DMSO</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Helper pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Manometer</td><td>Riester</td><td>LF1459</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W558818</td><td>75&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Or any equilavent product</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Video capture software</td><td>Hypercam</td><td>version 2.28.01</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Patch clamping</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2411</td><td>3 mg disolved daily in 50 &amp;mu;L of DMSO (sonicate to dissolve)</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200a</td><td>Or any equilavent product</td></tr><tr><td>Analogue digital converter</td><td>Axon</td><td>Digidata 1440A</td><td>Or any equilavent product</td></tr><tr><td>Collagenase Type 1A</td><td>Sigma-Aldrich</td><td>C9891&amp;nbsp;</td><td>0.1mg/mL in LCH</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>DNAse I</td><td>Millipore</td><td>260913</td><td>Working stock 1 MU mL-1 (10 MU diluted in 10 mL LCH)&amp;nbsp;</td></tr><tr><td>Faraday cage</td><td>Custom made</td><td></td><td>Any equilavent commerical product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclypse TE 300</td><td>Or any equilavent product</td></tr><tr><td>Microelectrode puller</td><td>Sutter instruments</td><td>P97</td><td>Or any equilavent product</td></tr><tr><td>Microforge +&amp;nbsp; Olympus CX 31 microscope</td><td>Glassworks</td><td>Fine Point F-550</td><td>Or any equilavent product</td></tr><tr><td>Micromanipulator</td><td>Sutter instruments</td><td>MP-285</td><td>Or any equilavent product</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Patching software</td><td>Molecular Devices</td><td>Pclamp v10.2</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>Or any equilavent product</td></tr><tr><td>Protease Type XIV</td><td>Sigma-Aldrich</td><td>P5147&amp;nbsp;</td><td>0.01mg/mL in LCH</td></tr><tr><td>Single channel pipette glass</td><td>World Percision instruments</td><td>IB150F-3</td><td>Or any equilavent product</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>USB camera</td><td>&amp;nbsp;Logitech&amp;nbsp;</td><td>HD Pro Webcam C920</td><td>Or any equilavent product</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>Whole cell pipette glass</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Or any equilavent product</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>x40 lens</td><td>Nikon</td><td>40x/0.55, WD 2.1, &amp;infin;/1.2</td><td>Or any equilavent product</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt; imaging</td><td></td><td></td><td></td></tr><tr><td>3-way taps</td><td>Cole-Parmer</td><td>UY-30600-02</td><td>Or any equilavent product</td></tr><tr><td>3-axis mechanical manipulator</td><td>Scientifica</td><td>LBM-7&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Acquisition software&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Acquisition Engine V1.1.5</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software for confocal imaging</td><td>Image J</td><td>https://downloads.imagej.net/fiji/</td><td>Free imaging software</td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7010</td><td>Or any equilavent product</td></tr><tr><td>Confocal microscope</td><td>Leica Geosystems</td><td>SP5&amp;nbsp;</td><td>Or any equilavent product; confocal</td></tr><tr><td>Digital Thermometer</td><td>RS</td><td>206-3722</td><td>Or any equilavent product</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>No. 7</td><td>Any superfine forcep</td></tr><tr><td>Glass bottomed recording chamber</td><td>Warner Instruments</td><td>64-0759</td><td>Or any equilavent product</td></tr><tr><td>In-line heater</td><td>Custom made</td><td></td><td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse TE2000</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Monochromator&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Optoscan</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</td><td>BD Plastipak</td><td>1mL</td><td>Syringe heated and pulled to internal diameter of pipette</td></tr><tr><td>Software for confocal microscope</td><td>Leica Geosystems</td><td>LAS-AF version 3.3.</td><td>Or any equilavent product; confocal</td></tr><tr><td>Suction pump</td><td>Interpet</td><td>AP1</td><td>Converted aeration pump by reversing bellows</td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>20mL Luer-lok</td><td>Or any equilavent product</td></tr><tr><td>Tungsten wire</td><td>Advent</td><td>W557418</td><td>50&amp;mu;m diameter</td></tr><tr><td>Tygon Tubing</td><td>VWR</td><td>miscellaneous sizes</td><td>Any equilavent product to fit</td></tr><tr><td>Water bath</td><td>Grant</td><td>Sub Aqua 12 Plus</td><td>Or any equilavent product</td></tr><tr><td>x100 lens</td><td>Nikon</td><td>x100 N.A. 1.3 oil</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Nikon</td><td>20x/0.40 WD 3.8, &amp;infin;/0.17</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x20 lens</td><td>Leica Geosystems</td><td>HCX PL FLUOTAR, 20x/0.50, &amp;infin;/0.17/D</td><td>Or any equilavent product; confocal</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x63 lens</td><td>Leica Geosystems</td><td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &amp;infin;/0.17/E</td><td>Or any equilavent product; confocal</td></tr><tr><td>Y-connectors</td><td>World Percision Instruments</td><td>14012</td><td>Or any equilavent product</td></tr><tr><td>&lt;strong&gt;Pipettes&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Specifications and settings for fabrication of pipettes for experimentation&lt;/strong&gt;</td></tr><tr><td>Helper pipette</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;10 M&amp;Omega;</td></tr><tr><td>Cannulation pipette</td><td>World Percision instruments</td><td>TW150F-4</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 290&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 58&lt;br/&gt; Time: 150&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 3-10 &amp;mu;m&lt;br/&gt; Polishing: no&lt;br/&gt; Final Resistance: &amp;lt;1 M&amp;Omega;</td></tr><tr><td>On-cell patching</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;5 M&amp;Omega;</td></tr><tr><td>Whole-cell patching</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 296&lt;br/&gt; Pressure: 200&lt;br/&gt; Heat: 287&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 50&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 2-3 &amp;mu;m&lt;br/&gt; Polishing: helpful but not necessary&lt;br/&gt; Final Resistance: 1-2 M&amp;Omega;</td></tr></tbody></table>

isolation of retinal arterioles for ex vivo cell physiology studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

isolation-of-retinal-arterioles-for-ex-vivo-cell-physiology-studies

Research

JoVE Journal

Biology

10.2K Views.  Queen's University of Belfast. This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies. 

Video: Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Neuroscience

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Medicine

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Novel Retina Protection Assay for Diabetic Retinopathy Pathogenesis Study

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

Author Spotlight: Understanding Retinal Vessel Resilience and Disease Progression

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

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Chapters in this video

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Purification of Mouse Brain Vessels

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice